Partial purification of biologically active, low molecular weight, human antihemophilic factor free of Von Willebrand factor. II. Further purification with thiol-disulfide interchange chromatography and additional evidence for disulfide bonds susceptible to limited reduction.
Thiol-disulfide interchange chromatography was used in the preparation of partially purified (approx. 17 000-fold) low molecular weight, Mr approximately or equal to 115 000, human antihemophilic factor essentially free of Von Willebrand factor. This antihemophilic factor was prepared from fresh plasma which had undergone limited reduction with 1 mM dithiothreitol and was subsequently reacted with 2,2'-dipyridyl disulfide, a sulfhydryl reagent which readily undergoes disulfide exchange. Exchange of protein-2-pyridyl mixed disulfide with thiopropyl-Sepharose resulted in the chromatographic adsorption of approx. 96% of the coagulant activity, of which approx. 20% subsequently eluted with 1.0 mM dithiothreitol. After reductive displacement from the thiopropyl-Sepharose the antihemophilic factor could be S-alkylated with iodo-[1-14]acetamide. The ratio of coagulant activity to Von Willebrand factor-antigen activity was greater than 30 000 : 1. In contrast, reduced antihemophilic factor was alkylated with iodoacetamide prior to chromatography as a control, and showed no exchange with the thiopropyl-Sepharose, eluting quantitatively in the breakthrough volume. These studies reinforce our previous results (Harris, R.B., Newman, J. and Johnson, A.J. (1981) Biochim. Biophys. Acta 668, 456-470) that partial reduction with dithiothreitol exposes critical sulfhydryl groups which, when alkylated, maintains the antihemophilic factor in a low molecular weight form without inactivating procoagulant activity.
D002845:Chromatography; D004220:Disulfides; D005169:Factor VIII / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds; D014841:von Willebrand Factor / Q000302:isolation & purification
D004220:Disulfides; D013438:Sulfhydryl Compounds; D014841:von Willebrand Factor; D005169:Factor VIII